Novel indazolylchromones: synthesis, fungicidal evaluation, molecular docking and aquatic toxicity prediction

Fungal diseases cause substantial loss to agricultural crops, affecting both quantities and quality. Although several methods are used for preventing disease incidence, fungicides remain crucial for higher yields and better quality. But in the past, the efficacy of several fungicides has decreased due to increased cases of fungicide resistant. In our pursuit of new effective fungicides, we synthesised a series of twenty 2-Indazol-1-yl-chromen-4-one derivatives (6a- 6t). The characterization of synthesized compounds was performed by several spectroscopic methods including Infrared, Nuclear Magnetic Resonance (1H and 13C) and HRMS. Out of 20 synthesised compounds, 19 (6b- 6t) were found to be novel. All synthesised indazolylchromones showed very good antifungal activity against Sclerotium rolfsii and Fusarium oxysporum. Among the tested compounds, 6t and 6f exhibited very good fungicidal activity against S. rolfsii with an ED50 of 10.10 mg L-1 and 16.18 mg L-1, respectively. In case of Fusarium oxysporum compound 6f displayed good’ activity with an ED50 value of 27.82 mg L-1. Molecular docking study was done to predict the binding sites of most active compounds, 6t and 6f with Cytochrome P450 14alpha -sterol demethylase (CYP51) enzyme using molsoft software. The acute toxicity predictions the of synthesized compounds for fish (LC50,96 Hr), daphnid (LC50, 48 Hr) and green algae (EC50, 96Hr) and the chronic toxicity predictions (ChV) were assessed using Ecological Structure Activity Relationship (ECOSAR) model. As per ECOSAR prediction, all the chemicals are inside AD and not missing predictions.


Introduction
Fungi represent one of the most harmful groups of phytopathogens, responsible for 80% of plant diseases (Agrios, 2005).Sclerotium rolfsii is a soil-borne fungus that generally occurs worldwide in hot and moderately warm places causing disease on hundreds of plant species.Typically, S. rolfsii attacks the plant near the surface of soil (Kator et al., 2015).The common diseases caused by S. rolfsii are foot rot, stem rot, root rot and wilt.Another important soil borne fungal pathogen is Fusarium oxysporum which causes fusarium wilt, a serious threat for agriculture (Fisher et al., 2012).F oxysporum is among the top ten most Synthesis of 2-Indazolyl-chromone (Gamill, 1979;Sugita et al., 1996;Kaushik et al., 2021).
Frontiers in Chemistry frontiersin.org02 Kundu et al. 10.3389/fchem.2024destructive fungal plant pathogens in the world (Dean et al., 2012).F oxysporum produces chlamydospores in the soil and survive for a longer time.It penetrates the roots, spread throughout the tissues, interfere with xylem and hinders waterflow resulting in wilting of plants.
Fungicides have been used for over two centuries for managing plant diseases, but in the recent past, many instances of fungicide resistance have led to the loss of many important fungicides (Hollomon, 2015).As a result, synthesis of new molecules has become increasingly important for crop protection.One of the approaches to address the problem of fungicide resistance is the development of hybrid fungicide molecules by combining different scaffolds.The hybrid molecules have multiple sites of action which minimizes the chances of fungicide resistance.
Azole fungicides have been widely used in agriculture are considered to be moderate risk fungicides for resistance development.Indazole is one of the important class of azoles.Indazoles are very rare in nature but a large number of synthetic indazole derivatives has been reported to have herbicidal activity (Hwang et al., 2005), insecticidal activity (Lahm et al., 2002), and fungicidal activity (Shakil et al., 2013;Tang et al., 2019).Thus, in the present study we synthesized indazolylchromones by combining the indazole moiety with chromones.

Chemicals and instruments
The chemicals required for synthesis of indazolylchromones were obtained from manufacturers.All the chemicals were utilized as received and not purified unless stated otherwise.The TLC plates (20 × 20 cm) coated with silica gel (having F254 fluorescence indicator) were used for reaction monitoring.100-200 mesh size silica gel was used for column chromatography.Melting points of synthesised compounds were measured by Buchi M-560, reported values were not adjusted/ corrected.NMR spectra of ( 1 H and 13 C) of synthesised compounds were recorded by JEOL JNM-ECZ400/SI.The mass spectrometry was carried out by ABSCIEX triple TOFTM 5600+ having Turbo Ion Spray.ED 50 values were estimated with the SPSS statistical package.Molecular docking was carried out using molsoft software.

Test fungus
Two fungal strains, F. oxysporum ITCC8113 and S. rolfsii ITCC 6866, were obtained from the Indian Type Culture Collection (ITCC) centre, Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, India.The strains were kept at a temperature of 27 °C for 4-7 days on Potato Dextrose Agar (PDA) slants.Before conducting the fungicidal bioassay, sub-culturing was performed on Petri plates.

In vitro fungicidal activity
A stock solution of each compound, with a concentration of 10,000 mg L -1 , was prepared using DMSO (Dimethylsulphoxide). From the stock solution, five different test concentrations were obtained through serial dilution, namely, 100 mg L -1 , 50 mg L -1 , 25 mg L -1 , 12.5 mg L -1 , and 6.25 mg L -1 for S. rolfsii and at 200 mg L -1 , 100 mg L -1 , 50 mg L -1 , 25 mg L -1 and 12.5 mg L -1 for F. oxysporum.Commerical available fungicide Hexaconazole 5% SC and Carbendazim 50% WP were used as positive control.
The fungicidal bioassay was conducted in a laboratory setting using the poisoned food technique (Nene et al., 1979).To determine the effectiveness of the antifungal treatment, the percentage of growth inhibition was computed using Abbott's formula (Abbott, 1925).
To calculate the corrected % inhibition (IC), the following formula was employed:  The ED 50 (mg L -1 ) values, which indicate the Effective Dose for 50% inhibition, were determined using the SPSS statistical package (v16.0).

Molecular docking
Molecular docking was carried out to predict the interaction of most active compounds, 6t and 6f with Cytochrome P450 14alpha -sterol demethylase (CYP51) enzyme using molsoft software and the respective docking scores were calculated to compare the interaction of compounds with the lanosterol 14αdemethylase (LDM) enzyme.

Aquatic toxicity prediction
The aquatic toxicity potentials of the synthesized compounds were assessed using Ecological Structure Activity Relationship (ECOSAR) model.It is a free and easy to use computer programme developed by US EPA.It predicts the median lethal concentration (LC 50 ), median effective concentration (EC 50 ), and chronic value (ChV) for various species.3D and 2D images of compound 6f with target protein CYP51.
Frontiers in Chemistry frontiersin.org08 3 Result and discussion

Synthesis and characterization of individual compounds
In the present work, total twenty indazolylchromones (6a-6t) were synthesized, out of which 19 compounds (6b-6t) were found novel.The indazolylchromones were synthesized by 4 steps synthetic scheme (scheme-I).In the last step, conjugated addition of 3-iodochromones 4a-4t with Indazole 5 is the Michael type addition reaction where indazole act as Michael donor (nucleophile) and chromone as Michael acceptor.In the first step indazole attacks chromone ring at 2-position and gives an intermediate A, which on removal of H-1 results in the formation of 2-(1-Indazolyl) chromones (Sugita et al., 1996).
Frontiers in Chemistry frontiersin.org10 1,532-1,537 (pyrone ring C=C stretch) supported the NMR data.The spectroscopic data revealed that in the above-synthesized compounds, the N atom at the one-position of the indazole was linked to chromone ring at C-2 position.

In vitro antifungal activity
All the synthesised indazolylchromones (6a-6t) exhibited antifungal activity against S. rolfsii (Table 2), but compound 6t (Figure 1) exhibited the highest activity (ED 50 = 10.10 mg L -1 ) and performed at par with that of Hexaconazole 5% SC (ED 50 = 8.57mg L - 1 ), a commercial fungicide.Among the alkoxy derivatives of indazolylchromones (6a-6n), butoxy derivative 6f, 2-Indazol-1-yl-7butoxy chromen-4-one, was found most active with ED 50 = 16.18 mg L - 1 .It was observed that alkoxy derivatives having even number of carbon chain length were more active as compared to odd number of carbon chain length.This trend was observed up to carbon chain length of C-6.After C-6 there was no regular trend was observed (Figure 2).

Molecular docking
The docking study of most potent compounds, 6f and 6t revealed that both compounds have different orientations, which in turn results in different modes of binding in the active site of CYP51.
In Figure 4, it has been observed that in compound 6f, the indazole ring is having π-π with TRY 76 and hydrophobic interactions with MET 79, PHE 255, PHE 78, VAL 434, LEU 321 residues.The chromone ring was found to have hydrophobic interactions with ALA 256 and HEM 460 residues.The butoxy chain of chromone ring was observed to be engaged in hydrophobic interactions with LYS 97, PHE 83, LEU 100 and ARG 96 residues.
In case of compound 6t (Figure 5), it has been observed that the indazole ring is having π-π with TRY 76 and hydrophobic interactions with PHE 78, LEU 321and HEM 460 residues.The chromone ring was found to have hydrophobic interactions with GLN 72, ALA 73, MET 79 THR 80 and PHE 83 residues.
In commercial fungicide hexaconazole (Figure 6), triazole ring showed hydrophobic interaction with HEM 460, HIS 259, THR 260 and ALA 256 residues.The benzyl ring was found to be engaged with PHE 255, HIE 101 PHE 83 and LEU 100 residues.

Aquatic toxicity
The acute toxicity predictions for fish (LC 50 ,96 Hr), daphnid (LC 50 , 48 Hr) and green algae (EC 50 , 96Hr) and the chronic toxicity predictions (ChV) were compared and tabulated in table 5. Fish LC 50 values varied from 1.64 × 10 −6 to 0.685 mg/L whereas the chronic toxicity values varied from 1.86 × 10 −4 to 0.042 mg/L among the different compounds, the highest toxicity was shown by the alkoxy substituted compounds.Among the alkoxy substituted compounds, the toxicity was found to be increasing with increase in the alkyl chain.Lowest toxicities were exhibited by compounds with halo substitutions.Among the halogenated compounds, dichlorinated compound exhibited the highest toxicity but less toxic as compared to alkoxy derivatives (Table 4).As per ECOSAR prediction, all the chemicals are inside AD and not missing predictions.It was observed that the acute as well as chronic toxicity values have negative linear correlations with the log K OW values (Figures 7,8).Plot of log ChV values against log K ow .
Frontiers in Chemistry frontiersin.org11 Kundu et al. 10.3389/fchem.2024.1411187In conclusion, a series of twenty compounds were synthesised and characterised successfully.Out of 20 compounds 19 were found novel (6b-6t).All the synthesized compounds were found to have fungicidal activity against S. rolfsii and F. oxysporum.It was observed that the synthesised compounds have less effectiveness against F. oxysporum.Compound 6t and 6f were found to be very effective against Sclerotium rolfsii.Also, molecular docking studies support the in vitro bioassay result where compound 6t with highest negative binding energy value (−17.77kcal mol −1 ) showed better binding with lanosterol enzyme.Therefore, these two compounds could be used for effective management of Sclerotium rolfsii.

I
denote the % inhibition, CF = (90 -C)/C x 100, where 90 is the Petriplate diameter (in mm) and C represents the growth of mycelium (in mm) in control.

FIGURE 3
FIGURE 3In vitro antifungal activity of 6f against Fusarium oxysporum.

FIGURE 5
FIGURE 53D and 2D images of compound 6t with target protein CYP51.

FIGURE 6
FIGURE 63D and 2D images of hexaconazole with target protein CYP51.

TABLE 2
In vitro antifungal activity of synthesized compounds against S. rolfsii.

TABLE 3
In vitro antifungal activity of synthesized compounds against F. oxysporum.

TABLE 4
Baseline toxicity of the synthesized compounds.